Pfcrt Haplotypes Research Articles

Haplotypes of Chloroquine Resistance Marker Genes Among Uncomplicated Malaria Cases in Lagos, Nigeria.

Drug resistance resulting from mutations in Plasmodium falciparum, that caused the failure of previously effective malaria drugs, has continued to threaten the global malaria elimination goal. This study describes the profiles of P. falciparum chloroquine resistance transporter (Pfcrt) and P. falciparum multidrug resistance 1 (Pfmdr1), the genetic markers associated with 4-aminoquinoline resistance, among P. falciparum isolates from Lagos, Nigeria. Genomic DNA was extracted from the dried blood spot samples obtained from individuals with microscopically confirmed P. falciparum infection in health facilities and communities in Lagos State, Nigeria. The DNA was amplified using nested polymerase chain reaction, and sequence analysis was performed to identify single nucleotide polymorphisms in the pfcrt and pfmdr1 genes. The study showed that 82.4% (178) of the isolates had pfmdr1 wild-type, while mutations were observed at codons N86Y (11.6%) and D1246Y (3.2%). Other mutations seen were at codons Y23S (0.5%), E130K (2.3%), and S149P (0.5%). 30.8% (64) of the isolates had pfcrt wild-type (CVMNK), while 62.0% (129) had CVIET (mutant) haplotype. Other pfcrt haplotypes detected include; CVIDT (1.9%); CVMDT (1.4%); CVIKT (1.0%); CVINT (0.5%); CVMET (0.5%); CVMKT (0.5%); CVMNT (1.0%); and CVMEK (0.5%). The findings underscore the presence of uncommon pfcrt haplotypes and a high prevalence of drug-resistant pfcrt haplotypes (CVIET), alongside a high prevalence of wild-type pfmdr in Lagos. This study highlights the need for ongoing surveillance of these genetic markers to provide data that can inform decisions on malaria case management and preserve the efficacy of artemisinin combination therapies (ACTs) in Nigeria.

Plasmodium falciparum African PfCRT Mutant Isoforms Conducive to Piperaquine Resistance are Infrequent and Impart a Major Fitness Cost.

Piperaquine, used in combination with dihydroartemisinin, has been identified as a promising partner drug for uncomplicated treatment and chemoprevention of Plasmodium falciparum malaria in Africa. In light of the earlier spread of piperaquine resistance in Southeast Asia, mediated primarily by mutations in the drug efflux transporter PfCRT, we have explored whether PfCRT mutations would represent a probable path to piperaquine resistance becoming established in Africa. We edited PfCRT mutations known to mediate piperaquine resistance in Southeast Asia into P. falciparum asexual blood stage parasites expressing three prevalent African mutant PfCRT haplotypes. Gene-edited clones were profiled in antimalarial concentration-response and competitive fitness assays. pfcrt-edited parasites expressing the contemporary Southeast Asian T93S or I218F mutations added to the GB4 and Cam783 haplotypes common in Africa did not mediate piperaquine resistance, with partial survival only at low drug concentrations. In contrast, parasites expressing these mutations on the rare PfCRT FCB haplotype, observed mostly in North-East Africa, acquired a moderate level of piperaquine resistance. Dd2GB4, Dd2Cam783, and Dd2FCB lines edited to express the T93S or I218F mutations showed increased susceptibility to chloroquine. Piperaquine-resistant African PfCRT isoforms conferred a substantial fitness cost, manifesting as reduced asexual blood stage parasite growth rates. These findings suggest that piperaquine-resistant PfCRT mutations that emerged in Southeast Asia mediate resistance only in a limited subset of African PfCRT haplotypes, with fitness costs that we suspect would likely preclude dissemination in high-transmission malaria-endemic African regions.

Genetic surveillance shows spread of ACT resistance during period of malaria decline in Vietnam (2018-2020).

Vietnam's goal to eliminate malaria by 2030 is challenged by the further spread of drug-resistant Plasmodium falciparum malaria to key antimalarials, particularly dihydroartemisinin-piperaquine (DHA-PPQ). The custom targeted NGS amplicon sequencing assay, AmpliSeq Pf Vietnam v2, targeting drug resistance, population genetic- and other markers, was applied to detect genetic diversity and resistance profiles in samples from 8 provinces in Vietnam (n = 354), in a period of steep decline of incidence (2018-2020). Variants in 14 putative resistance genes, including P. falciparum Kelch 13 (PfK13) and P. falciparum chloroquine resistance transporter (Pfcrt), were analyzed and within-country parasite diversity was evaluated. Other targets included KEL1-lineage markers and diagnostic markers of Pfhrp2/3. A concerning level of DHA-PPQ resistance was detected. The C580Y mutation in PfK13 was found in nearly 80% of recent samples, a significant rise from previous data. Vietnam has experienced a significant challenge with the spread of DHA-PPQ resistant malaria parasites, particularly in the provinces of Binh Phuoc and Gia Lai. Resistance spread to high levels in Binh Thuan prior to the country-wide treatment policy change from DHA-PPQ to pyronadine-artesunate (PA). A complex picture of PPQ-resistance dynamics was observed, with an increase of PPQ-resistance associated Pfcrt mutations, indicating an evolutionary response to antimalarial pressure. Additionally, the compensatory mutation C258W in Pfcrt, which increases chloroquine (CQ) resistance while reversing PPQ resistance, is emerging in Gia Lai following the adoption of PA as the first-line treatment. This study found high levels of multidrug resistance, with over 70% of parasites in 6 out of 8 provinces showing significant sulfadoxine-pyrimethamine (SP) resistance and widespread chloroquine-resistant Pfcrt haplotypes. We also report an absence of P. falciparum histidine rich protein 2 and 3 (Pfhrp2/3) gene deletions, ensuring the continued reliability of HRP2/3-based rapid diagnostic tests. P. falciparum populations in Vietnam are becoming more isolated, with clonal populations showing high geographical clustering by province. The central highlands, particularly Gia Lai province, have the highest residual malaria burden but exhibit low diversity and clonal populations, likely due to the pressures from the antimalarial drugs and targeted national malaria control program (NMCP) efforts. In conclusion, examining a broad panel of full-length resistance genes and SNPs provided high-resolution insights into genetic diversity and resistance evolution in Vietnam, offering valuable information to inform local treatment and intervention strategies.

Rapid and supersensitive allele detection of Plasmodium falciparum chloroquine resistance via a Pyrococcus furiosus argonaute-triggered dual-signal biosensing platform

BackgroundMalaria remains a serious public health problem worldwide, particularly in Africa. Resistance to antimalarial drugs is an essential issue for malaria control and elimination. Currently, polymerase chain reaction (PCR) combined with Sanger sequencing is regarded as the gold standard for mutation detection. However, this method fails to meet the requirements of point-of-care testing (POCT) because of its time-consuming, expensive instruments and professional dependence. To support this strategy, we developed a novel diagnostic platform that combines recombinase polymerase amplification (RPA) with the Pyrococcus furiosus argonaute (PfAgo) protein and was designed to detect gene mutations related to antimalarial drug resistance. The Pfcrt haplotypes CVMNK and CVIET of chloroquine resistance (CQR) were used as examples and were assessed in this study.MethodsBy meticulously designing strategies, RPA primers, guide DNAs, and probes were screened, the reaction was optimized, and the resulting parameters were employed to ascertain the genotype of Pfcrt. The recombinant plasmids pUC57/Pfcrt-CVIET and pUC57/Pfcrt-CVMNK were constructed and diluted for sensitivity detection. The pUC57/Pfcrt-CVIET plasmid mixture was added to the pUC57/Pfcrt-CVMNK plasmid mixture in different additions to configure several specific proportions of mixed plasmid mixtures. The RPA-PfAgo platform was used, and the mixed plasmid was detected simultaneously via nest-PCR (nPCR) and Sanger sequencing. The platform was then evaluated on 85 clinical samples and compared with Sanger sequencing.ResultsThe entire process achieves the key mutation Pfcrt-CVMNK/CVIET genotype identification of CQR within 90 min. The platform achieved 1.8 × 104 copies/μL sensitivity and could detect as little as 3% CVIET in mixed plasmids, which is a higher sensitivity than that of Sanger sequencing (5%). Notably, the platform shows 100% concordance with the gold standard method when 85 clinical samples are tested. The sensitivity and specificity were 100% for the 85 clinical samples.ConclusionsThis study established an RPA-PfAgo platform for genotyping the key mutation Pfcrt-CVMNK/CVIET of CQR. This method can rapidly produce reliable results and avoid the disadvantages of nPCR with sequencing. This approach has the characteristics of a short operation time, low device dependence, and a good match to the POCT strategy, suggesting that the platform can be easily applied locally or on site.Graphical abstract

Antimalarial drug resistance profiling of Plasmodium falciparum infections in India using Ion Torrent deep sequencing

IntroductionTracking the emergence and spread of antimalarial drug resistance is critical for supporting progress towards the control and eventual elimination of malaria in South Asia, especially India. Plasmodium falciparum has evolved resistance to virtually every antimalarial drug, and significant progress has been made to identify the molecular genetic mechanisms involved in the most common types of resistance.MethodsAn amplicon sequencing protocol was used for molecular surveillance of antimalarial drug resistance in a total of 158 patient isolates collected from December 2012 to September 2015 from three sites in south, west and east India: Tamil Nadu, Gujarat, and Odisha respectively. Five full length Plasmodium falciparum genes whose mutant proteins are implicated in antimalarial drug resistance were investigated: Pfcrt for chloroquine, Pfdhfr for pyrimethamine, Pfdhps for sulfadoxine, Pfk13 for artemisinin and Pfmdr1 for resistance to multiple antimalarial drugs.ResultsWe observed a high proportion of wild-type Pfcrt and Pfdhfr haplotypes from the P. falciparum-dominant site Rourkela, while mutant Pfcrt and Pfdhfr haplotypes were fixed at the P. vivax dominant sites Chennai and Nadiad. The wild-type Pfdhps haplotype was predominant across all study sites. We identified mutations in the propeller domain of Pfk13, although they are not associated with resistance to artemisinin. Finally, using samples taken from the same patient on day 2, day 7, and day 14 after artemisinin combination treatment, we were able to observe changes in allele frequency of drug resistance genes during the course of an infection.DiscussionThis is the first high-throughput deep sequencing study of five full-length P. falciparum genes in clinical isolates from three different study sites in India with varying transmission ecologies. Amplicon sequencing based on ion-torrent has the potential to track changes in the frequency of drug resistant alleles as a patient is undergoing drug therapy, and to identify new resistance mutations as they increase in frequency in the patient. This study showed possibility of whole gene sequencing, leads to in-depth molecular surveillance of multiple antimalarial resistant candidates and furthermore suggests investigations on reversal of resistant genotypes due to usage of artemisinin combination therapy in P. falciparum prevalent areas of the country.

Prevalence of polymorphisms in marker genes associated with antimalarial drug resistance in Plasmodium falciparum following 10 years of artemisinin-based combination therapy implementation in urban Kolkata.

Resistance to antimalarial drugs is one of the major challenges for malaria elimination. In India, artemisinin combination therapy (artesunate-sulfadoxin pyrimethamine) was introduced in place of chloroquine (CQ) for the treatment of uncomplicated falciparum malaria in 2010. Periodical monitoring of polymorphisms in antimalarial drug resistance marker genes will be useful for assessing drug pressure, mapping and monitoring of drug resistance status; and will be helpful for searching alternative treatments. This study was conducted to study the polymorphisms in antimalarial drug resistance marker genes among clinical Plasmodium falciparum isolates collected from Kolkata after 10 years of artemisinin-based combination therapie (ACT) implementation. Blood samples were collected from P. falciparum mono-infected patients and polymorphisms in P. falciparum CQ resistance transporter (pfcrt), P. falciparum multidrug resistance (pfmdr-1), P. falciparum dihydrofolate reductase (pfdhfr), P. falciparum dihydropteroate synthetase (pfdhps), pfATPase6 and pfK-13 propeller genes were analysed by polymerase chain reaction and DNA sequencing. In pfcrt gene, C72S, and K76T mutation was recorded in 100% isolates and no mutations was detected in any of the targeted codons of pfmdr-1 gene. A double mutant pfcrt haplotype SVMNT and wildtype haplotype NYD in pfmdr-1 were prevalent in 100% of study isolates. Triple mutant pfdhfr-pfdhps haplotype ANRNI-SGKAA was recorded. No polymorphism in pfK13 gene was documented in any of the isolates. Observed wild codon N86 along with Y184 and D1246 of pfmdr-1 gene might be an indication of the reappearance of CQ sensitivity. The absence of quadruple and quintuple haplotypes in pfdhfr-pfdhps gene along with the wild haplotype of pfK13 is evidence of ACT effectivity. Hence, similar studies with large sample size are highly suggested for monitoring the drug resistance status of P. falciparum.

Molecular markers associated with drug resistance in Plasmodium falciparum parasites in central Africa between 2016 and 2021.

The widespread occurrence of anti-malarial drug resistance threatens the current efforts to control malaria in African regions. Molecular marker surveillance helps to track the emergence and spread of drug-resistant malaria cases. A total of 237 Plasmodium falciparum infections imported from central Africa to Zhejiang Province, China, between 2016 and 2021, were investigated. Genomic DNA was extracted from blood samples of each patient and nested PCRs was used to detect molecular markers in k13, Pfcrt, and Pfmdr1 genes. The spatial and temporal distributions of the molecular markers were analyzed. A limited polymorphism of k13 was observed, including two nonsynonymous (D464E and K503E) and five synonymous mutations. Wild-type CVMNK of Pfcrt predominated (78.5%), whereas 19.5% of the samples harbored the mutant haplotype, CVIET. The point mutation Y184F and the single mutant haplotype NF of Pfmdr1 were the most frequently observed. The geographical distributions of the Pfcrt and Pfmdr1 haplotypes displayed distinct patterns, with the mutant haplotype of Pfcrt more common in Gabon (53.9%) and Congo (50.0%), and wild haplotypes of Pfmdr1 more frequently found in Cameroon, Angola, and Congo. The prevalence of wild-type CVMNK of Pfcrt increased from 68.5-74.6% in 2016-2017 to 81.8-87.5% in 2018-2021. The proportion of wild-type Pfmdr1 also increased from 27.1% in 2016 to 38.5% in 2019. The geographical and temporal distribution of k13, Pfcrt, and Pfmdr1 polymorphisms in P. falciparum parasites imported from central Africa between 2016 and 2021 are demonstrated. Our data provide updated evidence that can be used to adjust anti-malarial drug policies in central Africa and China.

Clinical isolates of uncomplicated falciparum malaria from high and low malaria transmission areas show distinct pfcrt and pfmdr1 polymorphisms in western Ethiopia

BackgroundPfcrt gene has been associated with chloroquine resistance and the pfmdr1 gene can alter malaria parasite susceptibility to lumefantrine, mefloquine, and chloroquine. In the absence of chloroquine (CQ) and extensive use of artemether–lumefantrine (AL) from 2004 to 2020 to treat uncomplicated falciparum malaria, pfcrt haplotype, and pfmdr1 single nucleotide polymorphisms (SNPs) were determined in two sites of West Ethiopia with a gradient of malaria transmission.Methods230 microscopically confirmed P. falciparum isolates were collected from Assosa (high transmission area) and Gida Ayana (low transmission area) sites, of which 225 of them tested positive by PCR. High-Resolution Melting Assay (HRM) was used to determine the prevalence of pfcrt haplotypes and pfmdr1 SNPs. Furthermore, the pfmdr1 gene copy number (CNV) was determined using real-time PCR. A P-value of less or equal to 0.05 was considered significant.ResultsOf the 225 samples, 95.5%, 94.4%, 86.7%, 91.1%, and 94.2% were successfully genotyped with HRM for pfcrt haplotype, pfmdr1-86, pfmdr1-184, pfmdr1-1042 and pfmdr1-1246, respectively. The mutant pfcrt haplotypes were detected among 33.5% (52/155) and 80% (48/60) of isolates collected from the Assosa and Gida Ayana sites, respectively. Plasmodium falciparum with chloroquine-resistant haplotypes was more prevalent in the Gida Ayana area compared with the Assosa area (COR = 8.4, P = 0.00). Pfmdr1-N86Y wild type and 184F mutations were found in 79.8% (166/208) and 73.4% (146/199) samples, respectively. No single mutation was observed at the pfmdr1-1042 locus; however, 89.6% (190/212) of parasites in West Ethiopia carry the wild-type D1246Y variants. Eight pfmdr1 haplotypes at codons N86Y–Y184F–D1246Y were identified with the dominant NFD 61% (122/200). There was no difference in the distribution of pfmdr1 SNPs, haplotypes, and CNV between the two study sites (P > 0.05).ConclusionPlasmodium falciparum with the pfcrt wild-type haplotype was prevalent in high malaria transmission site than in low transmission area. The NFD haplotype was the predominant haplotype of the N86Y–Y184F–D1246Y. A continuous investigation is needed to closely monitor the changes in the pfmdr1 SNPs, which are associated with the selection of parasite populations by ACT.

Fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum clinical isolates collected from unrest tribal agencies of Pakistan.

Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.

Attributable risk factors for asymptomatic malaria and anaemia and their association with cognitive and psychomotor functions in schoolchildren of north-eastern Tanzania.

In Africa, children aged 5 to 15 years (school age) comprises more than 50% (>339 million) of the under 19 years population, and are highly burdened by malaria and anaemia that impair cognitive development. For the prospects of improving health in African citizens, understanding malaria and its relation to anaemia in school-aged children, it is crucial to inform targeted interventions for malaria control and accelerate elimination efforts as part of improved school health policy. We conducted a study to determine the risk factors for asymptomatic malaria and their association to anaemia. We explored the prevalence of antimalarial drug resistance as well as the association of asymptomatic malaria infection and anaemia on cognitive and psychomotor functions in school-aged children living in high endemic areas. This study was a comprehensive baseline survey, within the scope of a randomised, controlled trial on the effectiveness and safety of antimalarial drugs in preventing malaria and its related morbidity in schoolchildren. We enrolled 1,587 schoolchildren from 7 primary schools located in Muheza, north-eastern Tanzania. Finger-pricked blood samples were collected for estimation of malaria parasitaemia using a microscope, haemoglobin concentration using a haemoglobinometer, and markers of drug resistance processed from dried blood spots (DBS). Psychomotor and Cognitive functions were assessed using a ‘20 metre Shuttle run’ and a test of everyday attention for children (TEA-Ch), respectively. The prevalence of asymptomatic malaria parasitaemia, anaemia and stunting was 26.4%, 49.8%, and 21.0%, respectively with marked variation across schools. In multivariate models, asymptomatic malaria parasitaemia attributed to 61% of anaemia with a respective population attribution fraction of 16%. Stunting, not sleeping under a bednet and illiterate parent or guardian were other factors attributing to 7%, 9%, and 5% of anaemia in the study population, respectively. Factors such as age group (10–15 years), not sleeping under a bednet, low socioeconomic status, parents’ or guardians’ with a low level of education, children overcrowding in a household, and fewer rooms in a household were significantly attributed to higher malaria infection. There was no significant association between malaria infection or anaemia and performance on tests of cognitive function (sustained attention) or psychomotor function (VO2 max). However, a history of malaria in the past one month was significantly associated with decreased cognitive scores (aOR = -4.1, 95% CI -7.7–0.6, p = 0.02). Furthermore, stunted children had significantly lower VO2max scores (aOR = -1.9, 95% CI -3.0–0.8, p = 0.001). Regarding the antimalarial drug resistance markers, the most prevalent Pfmdr1 86-184-1034-1042-1246 haplotypes were the NFSND in 47% (n = 88) and the NYSND in 52% (n = 98). The wild type Pfcrt haplotypes (codons 72–76, CVMNK) were found in 99.1% (n = 219) of the samples. Malaria, stunting and parents’ or guardians’ illiteracy were the key attributable factors for anaemia in schoolchildren. Given malaria infection in schoolchildren is mostly asymptomatic; an addition of interventional programmes such as intermittent preventive treatment of malaria in schoolchildren (IPTsc) would probably act as a potential solution while calling for an improvement in the current tools such as bednet use, school food programme, and community-based (customised) health education with an emphasis on nutrition and malaria control.

Molecular surveillance for polymorphisms associated with artemisinin-based combination therapy resistance in Plasmodium falciparum isolates collected in Mozambique, 2018

BackgroundDue to the threat of emerging anti-malarial resistance, the World Health Organization recommends incorporating surveillance for molecular markers of anti-malarial resistance into routine therapeutic efficacy studies (TESs). In 2018, a TES of artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ) was conducted in Mozambique, and the prevalence of polymorphisms in the pfk13, pfcrt, and pfmdr1 genes associated with drug resistance was investigated.MethodsChildren aged 6–59 months were enrolled in four study sites. Blood was collected and dried on filter paper from participants who developed fever within 28 days of initial malaria treatment. All samples were first screened for Plasmodium falciparum using a multiplex real-time PCR assay, and polymorphisms in the pfk13, pfcrt, and pfmdr1 genes were investigated by Sanger sequencing.ResultsNo pfk13 mutations, associated with artemisinin partial resistance, were observed. The only pfcrt haplotype observed was the wild type CVMNK (codons 72–76), associated with chloroquine sensitivity. Polymorphisms in pfmdr1 were only observed at codon 184, with the mutant 184F in 43/109 (39.4%) of the samples, wild type Y184 in 42/109 (38.5%), and mixed 184F/Y in 24/109 (22.0%). All samples possessed N86 and D1246 at these two codons.ConclusionIn 2018, no markers of artemisinin resistance were documented. Molecular surveillance should continue to monitor the prevalence of these markers to inform decisions on malaria treatment in Mozambique.

The return of chloroquine-sensitive Plasmodium falciparum parasites in Jazan region, southwestern Saudi Arabia over a decade after the adoption of artemisinin-based combination therapy: analysis of genetic mutations in the pfcrt gene.

This study investigated the polymorphism in the P. falciparum chloroquine resistance transporter (pfcrt) gene 11years after chloroquine (CQ) cessation in Jazan region, southwestern Saudi Arabia. Two hundred and thirty-five P. falciparum isolates were amplified to detect mutations in the pfcrt gene. The pfcrt 76T molecular marker for CQ resistance was detected in 66.4% (156/235) of the isolates, while the K76 CQ-sensitive wild type was detected in 33.6%. The pfcrt 74I and pfcrt 75E point mutations were each found to be present in 56.2% of isolates, while only four isolates (1.7%) were found to carry the pfcrt 72S mutation. Moreover, four pfcrt haplotypes were identified as follows: the CVIET triple-allele (56.2%), SVMET double-allele (1.7%) and CVMNT single-allele (8.5%) mutant haplotypes and the CVMNK wild haplotype (33.6%). The analysis also revealed significant associations between the prevalence of mutant pfcrt alleles and haplotypes and the age group, governorate and nationality of the patients as well as the parasitaemia level (p < 0.05). The findings provide evidence of the potential re-emergence of CQ-susceptible P. falciparum strains in Jazan region over a decade after CQ discontinuation, with about one third of the isolates analysed carrying the pfcrt K76 CQ-sensitive wild allele and the CVMNK ancestral wild haplotype. Although the reintroduction of CQ cannot be recommended at present in Saudi Arabia, these findings support the rationale for a potential future role for CQ in malaria treatment. Therefore, continuous molecular and in vitro monitoring mutations of pfcrt polymorphism in Jazan region is highly recommended.

Subpatent Plasmodium with mutant pfmdr1, pfcrt, and pvmdr1 alleles from endemic provinces in Mindanao, the Philippines: implications for local malaria elimination

ObjectivesThis study was performed to identify and characterize circulating Plasmodium species in three provinces of Mindanao approaching malaria elimination. MethodsRapid diagnostic tests (RDT), microscopic examination, and PCR were used to detect malaria parasites. PCR-positive isolates were genotyped for polymorphisms in loci of interest. ResultsA total of 2639 participants were surveyed in Mindanao between 2010 and 2013. Malaria prevalence by PCR was 3.8% (95% confidence interval (CI): 2.7–5.2%) in Sarangani, 10% (95% CI: 7.7–12.7%) in South Cotabato, and 4.2% (95% CI: 3.2–5.6%) in Tawi-Tawi. P. falciparum and P. vivax were identified in all three provinces, and there was one case of P. malariae in South Cotabato. RDT was inferior to PCR for detecting asymptomatic P. falciparum and P. vivax. In Tawi-Tawi, microscopy failed to identify 46 PCR-positive malaria infections. The presence of pfcrt haplotypes CVMNK, CVIET, and SMNT (codons 72–76), pfmdr1 haplotype NFSND (codons 86, 184, 1034, 1042, 1246), and pvmdr1 haplotype NFL (codons 91, 976, 1076) was confirmed in Mindanao. ConclusionsAsymptomatic Plasmodium infections persisted in local communities between 2010 and 2013. PCR successfully identified subpatent malaria infections, and can better characterize malaria epidemiology in communities seeking malaria control and elimination at the local level.

Molecular surveillance of anti-malarial resistance pfcrt, pfmdr1, and pfk13 polymorphisms in African Plasmodium falciparum imported parasites to Wuhan, China

BackgroundImported malaria parasites with anti-malarial drug resistance (ADR) from Africa is a serious public health challenge in non-malarial regions, including Wuhan, China. It is crucial to assess the ADR status in African Plasmodium falciparum isolates from imported malaria cases, as this will provide valuable information for rational medication and malaria control.MethodsDuring 2017–2019, a cross-sectional study was carried out in Wuhan, China. Peripheral blood 3 ml of returned migrant workers from Africa was collected. The target fragments from pfcrt, pfmdr1, and k13 propeller (pfk13) genes were amplified, sequenced, and analysed.ResultsIn total, 106 samples were collected. Subsequently, 98.11% (104/106), 100% (106/106), and 86.79% (92/106) of these samples were successfully amplified and sequenced for the pfcrt (72–76), pfmdr1, and pfk13 genes, respectively. The prevalence of the pfcrt 76 T, pfmdr1 86Y, and pfmdr1 184F mutations was 9.62, 4.72, and 47.17%, respectively. At codons 72–76, the pfcrt locus displayed three haplotypes, CVMNK (wild-type), CVIET (mutation type), CV M/I N/E K/T (mixed type), with 87.50%, 9.62%, and 2.88% prevalence, respectively. For the pfmdr1 gene, NY (wild type), NF and YF (mutant type), N Y/F, Y Y/F, and N/Y Y/F (mixed type) accounted for 34.91, 43.40, 3.77, 15.09, 0.94, and 1.89% of the haplotypes, respectively. A total of 83 isolates with six unique haplotypes were found in pfcrt and pfmdr1 combined haplotypes, of which NY-CVMNK and NF-CVMNK accounted for 40.96% (34/83) and 43.37% (36/83), respectively. Furthermore, 90 cases were successfully sequenced (84.91%, 90/106) at loci 93, 97, 101, and 145, and 78 cases were successfully sequenced (73.58%, 78/106) at loci 343, 353, and 356 for pfcrt. However, the mutation was observed only in locus 356 with 6.41%. For pfk13, mutations reported in Southeast Asia (at loci 474, 476, 493, 508, 527, 533, 537, 539, 543, 553, 568, 574, 578, and 580) and Africa (at loci 550, 561, 575, 579, and 589) were not observed.ConclusionsThe present data from pfcrt and pfmdr1 demonstrate that anti-malarial drugs including chloroquine, amodiaquine, and mefloquine, remain effective against malaria treatment in Africa. The new mutations in pfcrt related to piperaquine resistance remain at relatively low levels. Another source of concern is the artemether-lumefantrine resistance-related profiles of N86 and 184F of pfmdr1. Although no mutation in pfk13 is detected, molecular surveillance must continue.

Surveillance of Plasmodium falciparum pfcrt haplotypes in southwestern Uganda by high\u2010resolution melt analysis

Background Chloroquine (CQ) resistance is conferred by mutations in the Plasmodium falciparum CQ resistance transporter (pfcrt). Following CQ withdrawal for anti-malarial treatment, studies across malaria-endemic countries have shown a range of responses. In some areas, CQ sensitive parasites re-emerge, and in others, mutant haplotypes persist. Active surveillance of resistance mutations in clinical parasites is essential to inform treatment regimens; this effort requires fast, reliable, and cost-effective methods that work on a variety of sample types with reagents accessible in malaria-endemic countries.MethodsQuantitative PCR followed by High-Resolution Melt (HRM) analysis was performed in a field setting to assess pfcrt mutations in two groups of clinical samples from Southwestern Uganda. Group 1 samples (119 in total) were collected in 2010 as predominantly Giemsa-stained slides; Group 2 samples (125 in total) were collected in 2015 as blood spots on filter paper. The Rotor-Gene Q instrument was utilized to assess the impact of different PCR-HRM reagent mixes and the detection of mixed haplotypes present in the clinical samples. Finally, the prevalence of the wild type (CVMNK) and resistant pfcrt haplotypes (CVIET and SVMNT) was evaluated in this understudied Southwestern region of Uganda.ResultsThe sample source (i.e. Giemsa-stained slides or blood spots) and type of LCGreen-based reagent mixes did not impact the success of PCR-HRM. The detection limit of 10− 5 ng and the ability to identify mixed haplotypes as low as 10 % was similar to other HRM platforms. The CVIET haplotype predominated in the clinical samples (66 %, 162/244); however, there was a large regional variation between the sample groups (94 % CVIET in Group 1 and 44 % CVIET in Group 2).ConclusionsThe HRM-based method exhibits the flexibility required to conduct reliable assessment of resistance alleles from various sample types generated during the clinical management of malaria. Large regional variations in CQ resistance haplotypes across Southwestern Uganda emphasizes the need for continued local parasite genotype assessment to inform anti-malarial treatment policies.

Polymorphisms of pfcrt, pfmdr1, and K13-propeller genes in imported falciparum malaria isolates from Africa in Guizhou province, China

BackgroundImported falciparum malaria from Africa has become a key public health challenge in Guizhou Province since 2012. Understanding the polymorphisms of molecular markers of drug resistance can guide selection of antimalarial drugs for the treatment of malaria. This study was aimed to analyze the polymorphisms of pfcrt, pfmdr1, and K13-propeller among imported falciparum malaria cases in Guizhou Province, China.MethodFifty-five imported falciparum malaria cases in Guizhou Province during 2012–2016 were included in this study. Their demographic information and filter paper blood samples were collected. Genomic DNA of Plasmodium falciparum was extracted from the blood samples, and polymorphisms of pfcrt, pfmdr1, and K13-propeller were analyzed with nested PCR amplification followed by sequencing. Data were analyzed with the SPSS17.0 software.ResultsThe prevalence of pfcrt K76T, pfmdr1 N86Y, and pfmdr1 Y184F mutation was 56.6, 22.2, and 72.2%, respectively, in imported falciparum malaria cases in Guizhou Province. We detected two mutant haplotypes of pfcrt, IET and MNT, with IET being more commonly found (54.7%), and five mutant haplotypes of pfmdr1, of which NFD was the most frequent (53.7%). There were totally 10 combined haplotypes of pfcrt and pfmdr1, of which the haplotype IETNFD possessed a predominance of 28.8%. In addition, three nonsynonymous mutations (S459T, C469F, and V692L) and two synonymous mutations (R471R and V589V) were detected in K13-propeller, all having prevalence less than 6.0%. In particular, a candidate K13 resistance mutation, C469F, was identified for the first time from Democratic Republic of the Congo with the prevalence of 2.0%.ConclusionsThe high prevalence of IET haplotype of pfcrt and NFD haplotype of pfmdr1 suggests the presence of chloroquine, artemether/lumefantrine, and dihydroartemisinin/piperaquine resistance in these cases. Therefore cautions should be made to artemisinin therapy for P. falciparum in Africa. Continuous monitoring of anti-malarial drug efficacy in imported malaria cases is helpful for optimizing antimalarial drug therapy in Guizhou Province, China.

Molecular surveillance of Pfcrt and k13 propeller polymorphisms of imported Plasmodium falciparum cases to Zhejiang Province, China between 2016 and 2018

BackgroundResistance to anti-malarial drugs hinders malaria elimination. Monitoring the molecular markers of drug resistance helps improve malaria treatment policies. This study aimed to assess the distribution of molecular markers of imported Plasmodium falciparum infections.MethodsIn total, 485 P. falciparum cases imported from Africa, Southeast Asia, and Oceania into Zhejiang province, China, from 2016 to 2018 were investigated. Most were imported from Africa, and only a few cases originated in Asia and Oceania. Blood samples were collected from each patient. Plasmodium falciparum chloroquine resistance transporter (Pfcrt) at residues 72–76 and Kelch13-propeller (k13) were determined by nested PCR and DNA sequence.ResultsWild-type Pfcrt at residues 72–76 was predominant (72.61%), but mutant and mixed alleles were also detected, of which CVIET (22.72%) was the most common. Mutant Pfcrt haplotypes were more frequent in patients from West Africa (26.92%), North Africa (25%), and Central Africa (21.93%). The number of cases of P. falciparum infections was small in Southeast Asia and Oceania, and these cases involved Pfcrt mutant type. For the k13 propeller gene, 26 samples presented 19 different point mutations, including eight nonsynonymous mutations (P441S, D464E, K503E, R561H, A578S, R622I, V650F, N694K). In addition, R561H, one of the validated SNPs in k13, was detected in one patient from Myanmar and one patient from Rwanda. A578S, although common in Africa, was found in only one patient from Cameroon. R622I was detected in one sample from Mozambique and one sample from Somalia. The genetic diversity of k13 was low in most regions of Africa and purifying selection was suggested by Tajima’s D test.ConclusionsThe frequency and spatial distributions of Pfcrt and k13 mutations associated with drug resistance were determined. Wild-type Pfcrt was dominant in Africa. Among k13 mutations correlated with delayed parasite clearance, only the R561H mutation was found in one case from Rwanda in Africa. Both Pfcrt and k13 mutations were detected in patients from Southeast Asia and Oceania. These findings provide insights into the molecular epidemiological profile of drug resistance markers in the study region.

Molecular epidemiology and evolution of drug-resistant genes in the malaria parasite Plasmodium falciparum in southwestern Nigeria

Malaria is an age-old disease of human kind living in the tropical and sub-tropical regions of the globe, with Africa contributing the highest incidence of morbidity and mortality. Among many hurdles, evolution and spread of drug-resistant Plasmodium falciparum parasites constitute major challenges to malaria control and elimination. Information on molecular epidemiology and pattern of evolution of genes conferring resistance to different antimalarials are needed to track the route of the spread of resistant parasites and also to inform if the drug-resistant genes are adapted in the population following the Darwinian model of evolution. In the present study, we have followed molecular methods to detect both the known and emerging mutations in three genes (Pfcrt, Pfdhfr and Pfdhps) of P. falciparum conferring resistance to chloroquine and sulfadoxine-pyrimethamine from two different states (Edo: meso-endemic and Lagos: hypo-endemic) in southwestern Nigeria. High diversities in haplotypes and nucleotides in genes responsible for chloroquine (Pfcrt) and sulfadoxine (Pfdhps) resistance are recorded. About 96% of Pfdhfr and Pfdhps gene in both the meso- and hypo- endemic areas were mutant type, followed by 61% in Pfcrt gene. Many unique haplotypes of Pfdhps and Pfcrt were found to be segregated in these two populations. One particular mutant haplotype of Pfdhfr (AIRNI) was found to be in very high frequency in both Lagos and Edo. While the net haplotype diversity was highest in Pfdhps (0.81 in Lagos, 0.87 in Edo), followed by Pfcrt (0.69 in Lagos, 0.65 in Edo); highest number of haplotype was found in Pfdhps with 13 distinct haplotypes, followed by seven in Pfcrt and four in Pfdhfr gene. Moreover, detection of strong linkage among mutations of Pfcrt and Pfdhfr and feeble evidence for balancing selection in Pfdhps are indicative of evolutionary potential of mutation in genes responsible for drug resistance in Nigerian populations of P. falciparum.

Multiple Novel Mutations in Plasmodium falciparum Chloroquine Resistance Transporter Gene during Implementation of Artemisinin Combination Therapy in Thailand.

Mutations in the chloroquine resistance transporter gene of Plasmodium falciparum (Pfcrt) are associated with drug susceptibility status of chloroquine and other antimalarials that interfere with heme detoxification process including artemisinin. We aim to investigate whether an increase in duration of artemisinin combination therapy (ACT) in Thailand could affect mutations in Pfcrt. The complete coding sequences of Pfcrt and dihydrofolate reductase (Pfdhfr), and size polymorphisms of the merozoite surface proteins-1 and 2 (Pfmsp-1 and Pfmsp-2) of 189 P. falciparum isolates collected during 1991 and 2016 were analyzed. In total, 12 novel amino acid substitutions and 13 novel PfCRT haplotypes were identified. The most prevalent haplotype belonged to the Dd2 sequence and no wild type was found. A significant positive correlation between the frequency of Pfcrt mutants and the year of sample collection was observed during nationwide ACT implementation (r = 0.780; P = 0.038). The number of haplotypes and nucleotide diversity of isolates collected during 3-day ACT (2009-2016) significantly outnumbered those collected before this treatment regimen. Positive Darwinian selection occurred in the transmembrane domains only among isolates collected during 3-day ACT but not among those collected before this period. No remarkable change was observed in the molecular indices for other loci analyzed when similar comparisons were performed. An increase in the duration of artesunate in combination therapy in Thailand could exert selective pressure on the Pfcrt locus, resulting in emergence of novel variants. The impact of these novel haplotypes on antimalarial susceptibilities requires further study.

Surveillance of Antimalarial Resistance Pfcrt, Pfmdr1, and Pfkelch13 Polymorphisms in African Plasmodium falciparum imported to Shandong Province, China

Antimalarial drug resistance is a major public health problem in China. From 2012 to 2015, more than 75% of malaria cases in Shandong Province were P. falciparum returned from Africa. However, molecular marker polymorphisms of drug resistance in imported P. falciparum cases have not been evaluated. In this study, we analyzed polymorphisms of the Pfcrt, Pfmdr1, and Pfkelch13 genes in 282 P. falciparum cases returned from Africa to Shandong between 2012 and 2015. Among the isolates, polymorphisms were detected in codons 74–76 of Pfcrt and 86, 184, 1246 of Pfmdr1, among which K76T (36.6%) and Y184F (60.7%) were the most prevalent, respectively. Six Pfcrt haplotypes and 11 Pfmdr1 haplotypes were identified and a comparison was made on the prevalence of haplotypes among East Africa, West Africa, Central Africa and South Africa. One synonymous and 9 nonsynonymous mutations in Pfkelch13 were detected in the isolates (4.6%), among which a candidate artemisinin (ART) resistance mutation P553L was observed. The study establishes fundamental data for detection of chloroquine resistance (CQR) and ART resistance with molecular markers of the imported P. falciparum in China, and it also enriches the genetic data of antimalarial resistance for the malaria endemic countries in Africa.